The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef has no known catalytic activity, and interacts with multiple host cell signaling proteins, including members of the Src protein-tyrosine kinase family. Previous work from our laboratory has shown that Nef binds and activates Hck, a Src family kinase (SFK) selectively expressed in macrophages, which are an essential HIV target cell and viral reservoir. Other work has established that knock-down of Hck expression blocks M-tropic HIV replication in primary human macrophages, and that homozygous-null hck mice show longer latency for Nef-induced AIDS-like disease. These studies strongly implicate Nef-Hck protein complexes in AIDS pathogenesis and as rational targets for anti-HIV drug discovery. Recently, we developed an in vitro fluorescence-based assay to identify inhibitors of a homogeneous Nef:Hck complex, and have validated this assay in 384-well plates. In a preliminary screen of a small library of compounds biased towards kinase inhibitors, we obtained three confirmed hits, all of which exhibited activity in a secondary assay against HIV replication. We believe that this assay is ideal for use in the Molecular Libraries Screening Center Network (MLSCN), and propose the following Specific Aims to be conducted in conjunction with the Center: Aim 1 - Transfer and implement our Nef:Hck HTS assay in the MLSCN; Aim 2 - Validate hits obtained in the primary screen in secondary assays with Hck and other SFKs in the presence and absence of Nef; Aim 3 - Evaluate the cytotoxicity and preliminary anti-HIV activity of the confirmed hits. Due to the emergence of HIV strains resistant to conventional anti-retroviral agents, there is an urgent need to identify and validate new HIV targets for the development of novel therapeutics. HIV accessory proteins in general, and the Nef protein in particular, have been suggested as possible therapeutic targets but to date no useful compounds have been reported that work via these proteins. Our hypothesis, supported by preliminary data, is that HIV Nef in complex with a host cell kinase (Hck) essential for disease progression represents a rational target for novel anti-HIV drug discovery. Our published work strongly suggests that Interaction with Nef may induce a novel conformation of Hck that may be addressable with small molecules. In addition, our assay has the potential to identify novel structures that interact directly with Nef itself, compromising its ability to recruit and activate Hck in the assay. Performing a screen in conjunction with the MLSCN is a very exciting prospect because of the wide range of novel structures available. In addition to the potential for new therapeutics, identification of compounds that selectively modify HIV Nef function will be invaluable tools to explore the function of this enigmatic protein. [unreadable] [unreadable] [unreadable]